CN110536898B - Peptides and methods for treating diabetes - Google Patents

Abstract

The present invention relates to peptides such as HCPYCSLQPLALEGSLQKRG and their use in the treatment of type 1 diabetes and the generation of cytolytic cd4+ T cells.

Description

Peptides and methods for treating diabetes

Background

Several strategies have been described to prevent the generation of unwanted immune responses against antigens. WO2008/017517 describes a new strategy using peptides comprising MHC class II antigens of a given antigen protein and an oxidoreductase motif. These peptides convert cd4+ T cells into cell types with cytolytic properties, termed cytolytic cd4+ T cells (cytolytic cd4+ T cells). These cells are able to kill those Antigen Presenting Cells (APCs) that present the antigen from which the peptide is derived by triggering apoptosis. WO2008/017517 demonstrates this concept for allergic and autoimmune diseases (e.g. type I diabetes). In this context, insulin may act as a self antigen.

WO2009101207 and Carlier et al (2012) plosone 7,10e45366 further describe antigen specific cytolytic cells in more detail.

WO2009101206 describes the use of peptides having an oxidoreductase motif and a MCH class II epitope of a soluble alloantigen to prevent an immune response against such antigen when used in replacement therapy (e.g. an unwanted immune response against insulin injection in diabetics).

WO2016059236 discloses further modified peptides wherein additional histidines are present in the vicinity of the oxidoreductase motif.

In designing peptides for type I diabetes, many factors may be considered, such as the type of autoantigen (insulin, GAD 65, …), the specific domains and epitopes of the autoantigen, the oxidoreductase motif, the length between the oxidoreductase motif and the epitope sequence, and the amino acid sequence.

Disclosure of Invention

The present invention provides novel peptides derived from insulin for use in the treatment of type 1 diabetes.

Compared with the peptide of the prior art, the peptide of the invention has the advantages that: cytolytic CD4+ T cells that have been generated using these peptides have increased IFN-gamma and sFasL production. It is also believed that the production of granzyme B in the cd4+ T cells increases.

The increased expression levels of these markers indicate that the peptides of the invention have a greater capacity to produce cytolytic cd4+ T cells than prior art peptides.

One aspect of the invention relates to peptides of 12 to 50 amino acids in length comprising the tetrapeptide sequence Cxx [ CST ] [ SEQ ID No. 1] or [ CST ] xxC [ SEQ ID No. 2] (i.e. redox motif) and the sequence LALEGSLQK [ SEQ ID No. 3] (i.e. epitope) spaced 0 to 5 amino acids, preferably 0 to 4 amino acids apart (i.e. linker apart) from the tetrapeptide.

Embodiments of these peptides comprise the sequence Ccxx [ CST ] SLQPLALEGSLQK [ SEQ ID NO:4] or [ CST ] xxCSLQPLALLEGSLQK [ SEQ ID NO:5].

Other embodiments of these peptides comprise the sequence CxCSLQPLALLEGSLQK [ SEQ ID NO:6].

Other embodiments of these peptides comprise the sequences HCxx [ CST ] SLQPLALEGSLQK [ SEQ ID NO:7] or H [ CST ] xxCSLQPL ALEGSLQK [ SEQ ID NO:8].

Other embodiments of these peptides comprise the sequence HCxxCSLQPLALEGESLQK [ SEQ ID NO:9].

Other embodiments of these peptides comprise the Ccxx [ CST ] [ SEQ ID NO:1] or [ CST ] xxC [ SEQ ID NO:2] redox motif sequence and sequence SLQPLALEGSLQKRG [ SEQ ID NO:20].

Specific embodiments of these peptides consist of the following sequences:

Cxx[CST]SLQPLALEGSLQK[SEQ ID NO:4]、

[CST]xxCSLQPLALEGSLQK[SEQ ID NO:5]、

CxxCSLQPLALEGSLQK[SEQ ID NO:6]、

HCxx[CST]SLQPLALEGSLQK[SEQ ID NO:7]、

h [ CST ] xxCSLQPLALLEGSLQK [ SEQ ID NO:8] or

HCxxCSLQPLALEGSLQK[SEQ ID NO:9]。

Other embodiments of these peptides consist of the following sequences:

Cxx[CST]SLQPLALEGSLQKRG[SEQ ID NO:10]、

[CST]xxCSLQPLALEGSLQKRG[SEQ ID NO:11]、

CxxCSLQPLALEGSLQKRG[SEQ ID NO:12]、

HCxx[CST]SLQPLALEGSLQKRG[SEQ ID NO:13]、

h [ CST ] xxCSLQPLAEGSLQKRG [ SEQ ID NO:14] or

HCxxCSLQPLALEGSLQKRG[SEQ ID NO:15]。

In particular embodiments of these sequences, cxx [ CST ] [ SEQ ID NO:1] is CPY [ CST ] [ SEQ ID NO:16], [ CST ] xxC [ SEQ ID NO:2] is [ CST ] PYC [ SEQ ID NO:17], more particular CxC [ SEQ ID NO:18] is CPYC [ SEQ ID NO:19].

One specific embodiment is peptide HCPYCSLQPLALEGSLQKRG [ SEQ ID NO:26].

In the above embodiments, the redox motif is located on the N-terminal side of the epitope.

In another set of embodiments, the peptide has a redox motif on the C-terminal side of the epitope.

Another aspect of the invention relates to any of the peptides as disclosed above for use as a medicament, in particular for the treatment or prevention of type 1 diabetes or for alleviating the symptoms of type 1 diabetes.

Another aspect relates to a pharmaceutical composition comprising any of the peptides as disclosed above and a pharmaceutically acceptable carrier.

Another aspect relates to an in vitro method for generating a cytolytic cd4+ T cell population for APCs presenting insulin epitopes, comprising the steps of:

-providing peripheral blood cells;

-contacting said cells in vitro with any one of the immunogenic peptides as disclosed above; and

-expanding the cells in the presence of IL-2.

Another aspect relates to a cell population of cytolytic cd4+ T cells for insulin-presenting APCs obtainable by the above method for use as a medicament.

Another aspect relates to a population of cells obtainable by the above method for the treatment or prevention of type 1 diabetes.

Drawings

Fig. 1: release of sFasL in cell lines produced with p17-003 or p 17-001.

P17-003 or p17-001 specific cells (IL 5 positive cells; black histogram) were further stimulated with antigen presenting cells loaded with the peptides p17-003 or p17-001 for 24 hours (depending on the peptides used for their production), and the supernatant was collected after co-culture for 24 hours. IL-5 negative cells were used for control PBMC populations (open histogram). The results represent the mean +/-SD of sFasL concentrations corrected for cell number.

Fig. 2: the figure shows the response of the cell lines to stimulation of p17-003 or p17-001 in terms of cytokine production.

Detailed Description

The present invention will be described with respect to particular embodiments, but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The following terms or definitions are provided only to aid in understanding the present invention. Unless defined otherwise herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art of the present invention. The definitions provided herein should not be construed to have a scope less than that understood by one of ordinary skill in the art.

As will be clear to a person skilled in the art, all methods, steps, techniques and operations not specifically described may and have been performed in a manner known per se, unless otherwise indicated. For example, reference is again made to the standard handbook, the general background art mentioned above and other references cited therein.

As used herein, the singular forms "a", "an" and "the" include singular and plural referents unless the context clearly dictates otherwise. The term "any" when used in relation to an aspect, claim or embodiment as used herein refers to any single (i.e., any) and all combinations of the recited aspects, claims or embodiments.

The terms "comprises," "comprising," and "contains" as used herein are synonymous with "including," "comprising," or "containing," and are inclusive or open-ended, and do not exclude additional unrecited members, elements, or method steps. The term also encompasses embodiments "consisting essentially of …" and "consisting of …".

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that corresponding range, and the endpoints recited.

When referring to measurable values such as parameters, amounts, time intervals, etc., the term 'about' as used herein is intended to encompass variations of the specified value of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, more preferably +/-0.1% or less, so long as such variations are suitable for implementation in the disclosed invention. It is to be understood that the value itself, as referred to by the modifier 'about', is also specifically and preferably disclosed.

As used herein, the term "for" as used in "a composition for treating a disease" shall also disclose a corresponding method of treatment and corresponding use of the formulation for the manufacture of a medicament for treating a disease.

The term "peptide" as used herein refers to a molecule comprising an amino acid sequence of 12 to 200 amino acids linked by peptide bonds, but which may comprise non-amino acid structures.

The peptides according to the invention may contain any of the conventional 20 amino acids or modified forms thereof, or may contain non-naturally occurring amino acids incorporated by chemical peptide synthesis or by chemical or enzymatic modification.

The term "antigen" as used herein refers to the structure of a macromolecule, typically a protein (with or without a polysaccharide), or made from a protein composition comprising one or more haptens and comprising T cell epitopes.

The term "antigenic protein" as used herein refers to a protein comprising one or more T cell epitopes. Autoantigen or autoantigen protein as used herein refers to a human or animal protein present in the body that elicits an immune response in the same human or animal body.

The term "epitope" refers to one or several parts of an antigen protein (which may define conformational epitopes), which are specifically recognized and bound by antibodies or parts thereof (Fab ', fab2', etc.) or receptors present on the cell surface of B or T cell lymphocytes, and which are capable of inducing an immune response by said binding.

In the context of the present invention, the term "T cell epitope" refers to a dominant, sub-dominant or minor T cell epitope, i.e. a portion of an antigenic protein, specifically recognized and bound by a receptor on the cell surface of a T lymphocyte. Whether an epitope is dominant, sub-dominant or secondary depends on the immune response elicited against the epitope. Among all possible protein T cell epitopes, dominant depends on how frequently T cells recognize these epitopes and are able to activate them.

T cell epitopes are epitopes recognized by MHC class II molecules, consisting of a sequence of +/-9 amino acids, which fit into the groove of MHC class II molecules. In the peptide sequence representing a T cell epitope, the amino acids in the epitope are numbered P1 to P9, the amino acid N-terminal of the epitope is numbered P-1, P-2, etc., and the amino acid C-terminal of the epitope is numbered P+l, P+2, etc. Peptides recognized by MHC class II molecules, but not MHC class I molecules, are referred to as MHC class II restricted T cell epitopes.

The term "MHC" refers to "major histocompatibility antigen". In humans, MHC genes are referred to as HLA ("human leukocyte antigen") genes. Although not consistently followed, some documents use HLA to refer to HLA protein molecules and MHC to refer to genes encoding HLA proteins. Thus, the terms "MHC" and "HLA" are equivalent when used herein. The HLA system in humans has its equivalent, the H2 system, in mice. The most deeply studied HLA genes are the 9 so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQB 1, HLA-DRA and HLA-DRB1. In humans, MHC is divided into three regions: I. class II and III. A. The B and C genes belong to MHC class I, while the 6D genes belong to class II. MHC class I molecules consist of a single polymorphic chain containing 3 domains (α1, 2 and 3) associated with β2 microglobulin on the cell surface. Class II molecules consist of 2 polymorphic chains, each containing 2 chains (α1 and 2, and β1 and 2).

Class I MHC molecules are expressed on almost all nucleated cells.

Peptide fragments presented in the context of class I MHC molecules are recognized by cd8+ T lymphocytes (cytolytic T lymphocytes or CTLs). Cd8+ T lymphocytes often mature into cytolytic effectors that lyse cells bearing the stimulating antigen. Class II MHC molecules are expressed predominantly on activated lymphocytes and antigen presenting cells. Cd4+ T lymphocytes (helper T lymphocytes or Th) are activated by recognition of unique peptide fragments presented by MHC class II molecules, which are typically found on antigen presenting cells such as macrophages or dendritic cells. CD4+ T lymphocytes proliferate and secrete cytokines such as IL-2, IFN-gamma, and IL-4, supporting antibody-mediated and cell-mediated responses.

Functional HLA is characterized by deep binding grooves to which endogenous as well as exogenous potentially antigenic peptides bind. The groove is also characterized by a well-defined shape and physicochemical properties. The HLA class I binding site is blocked because the peptide ends are fixed at the ends of the groove. They also involve hydrogen bonding networks with conserved HLA residues. In view of these limitations, the length of the binding peptide is limited to 8, 9 or 10 residues. However, peptides of up to 12 amino acid residues have also been shown to be able to bind HLA class I. Structural comparisons of different HLA complexes confirm the general binding pattern, where the peptide adopts a relatively linear extended conformation, or the central residue can be made to protrude from the groove.

In contrast to HLA class I binding sites, class II sites are open at both ends. This allows the peptide to extend from the actual binding region, thus "hanging" at both ends. Thus, HLA class II can bind peptide ligands of variable length ranging from 9 to more than 25 amino acid residues. Similar to HLA class I, the affinity of class II ligands is determined by the "constant" and "variable" components. The constant portion again results from the hydrogen bond network formed between the conserved residues in the HLA class II groove and the backbone of the binding peptide. However, this hydrogen bonding pattern is not limited to the N-and C-terminal residues of the peptide, but is distributed throughout the chain. The latter is important because it limits the conformation of the complex peptide to a strictly linear binding mode. This is common to all class II allotypes. The second component, which determines the binding affinity of the peptide, is variable due to certain positions of the polymorphism within the class II binding site. Different allotypes form different complementary pockets within the groove, thus addressing subtype-dependent selection or specificity of peptides. Importantly, the restriction on amino acid residues held in class II pockets is generally "softer" than class I. The cross-reactivity of peptides is much higher in different HLA class II allotypes. The +/-9 amino acid (i.e., 8, 9 or 10) sequences suitable for MHC class II T cell epitopes in the groove of MHC class II molecules are generally numbered P1 through P9. The N-terminal numbers of the additional amino acids of the epitope are P-1, P-2 and the like, and the C-terminal numbers of the amino acids of the epitope are P+1, P+2 and the like.

The term "homolog" as used herein with reference to an epitope as used in the context of the present invention refers to a molecule having at least 50%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% amino acid sequence identity to a naturally occurring epitope, thereby preserving the ability of the epitope to bind to an antibody or a cell surface receptor of a B and/or T cell. Specific homologs of the epitope correspond to the native epitope modified in at most three, more particularly at most 2, most particularly in one amino acid.

The term "derivative" as used herein with reference to the peptides of the invention refers to molecules containing at least the active part of the peptide (i.e. the redox motif and MHC class II epitope capable of eliciting cytolytic cd4+ T cell activity) and in addition to complementary parts which may have different purposes such as stabilizing the peptide or altering the pharmacokinetic or pharmacodynamic properties of the peptide.

The term "sequence identity" of two sequences as used herein relates to the number of positions having the same nucleotide or amino acid divided by the number of nucleotides or amino acids in the shorter sequence when the two sequences are aligned. In particular, the sequence identity is 70% to 80%,81% to 85%,86% to 90%,91% to 95%,96% to 100%, or 100%.

The terms "peptide-encoding polynucleotide (or nucleic acid)" and "peptide-encoding polynucleotide (or nucleic acid)" as used herein refer to a nucleotide sequence that, when expressed in a suitable environment, results in the production of the relevant peptide sequence or derivative or homologue thereof. Such polynucleotides or nucleic acids include normal sequences encoding the peptides, as well as derivatives and fragments of these nucleic acids that are capable of expressing peptides having the desired activity. A nucleic acid encoding a peptide or fragment thereof according to the invention is a sequence encoding a peptide or fragment thereof (most particularly a human peptide fragment) derived from or corresponding to a mammal.

The term "immune disorder" or "immune disease" refers to a disease in which the immune system's response is responsible for or maintains a dysfunctional or non-physiological condition in an organism. Immune disorders include, inter alia, allergic diseases and autoimmune diseases.

The term "autoimmune disease" or "autoimmune disorder" refers to a disease resulting from an abnormal immune response of an organism to its own cells and tissues due to the organism failing to recognize its own components (down to the sub-molecular level) as "self. This group of diseases can be divided into two categories, organ-specific diseases and systemic diseases. An "allergen" is defined as a substance, typically a macromolecular or protein composition, capable of eliciting lgE antibodies in a subject (atopic) susceptible, in particular genetically treated, patient. A similar definition is set forth in Liebers et al (1996) Clin. Exp. Allergy 26, 494-516.

The term "type 1 diabetes" (T1D) or "type 1 diabetes" (also known as "type 1 diabetes" or "immune-mediated diabetes" or previously known as "juvenile onset diabetes" or "insulin dependent diabetes") is an autoimmune disorder, typically developed in childhood susceptible individuals. On the basis of T1D, the pathogenesis is destruction of most insulin-producing pancreatic beta cells by autoimmune mechanisms. Briefly, organisms lose immune tolerance to pancreatic beta cells responsible for insulin production and induce an immune response, primarily cell-mediated, associated with autoantibody production, resulting in self-destruction of beta cells.

The term "therapeutically effective amount" refers to an amount of a peptide or derivative thereof of the present invention that produces a desired therapeutic or prophylactic effect in a patient. For example, when referring to a disease or disorder, it is an amount that reduces one or more symptoms of the disease or disorder to some extent, more specifically, it partially or completely restores to normal physiological or biochemical parameters associated with or that lead to the disease or disorder. Generally, a therapeutically effective amount is an amount of a peptide or derivative thereof of the invention that will result in an improvement or restoration of normal physiological conditions. For example, when used to treat a mammal affected by an immune disorder, it is daily amounts of peptide per kg of body weight of the mammal. Alternatively, in the case of administration by gene therapy, the amount of naked DNA or viral vector is adjusted to ensure local production of the relevant dose of the peptide, derivative or homologue of the invention.

When referring to peptides, the term "natural" refers to the fact that: the sequence is identical to a fragment of a naturally occurring protein (wild type or mutant). In contrast, the term "artificial" refers to sequences that do not exist in nature. Artificial sequences are obtained from natural sequences by limited modifications, such as altering/deleting/inserting one or more amino acids in the naturally occurring sequence, or by adding/removing amino acids at the N-or C-terminus of the naturally occurring sequence.

Amino acids are referred to herein by their full name, their three-letter abbreviation or their one-letter abbreviation.

The motifs of amino acid sequences are described herein according to the Prosite format. Motifs are used to describe certain sequence variants of specific parts of a sequence. The symbol X is used to accept any amino acid position. For a given position, a surrogate is indicated by listing acceptable amino acids between brackets (' [ ]). For example: [ CST ] represents an amino acid selected from Cys, ser or Thr. Amino acids that are alternatives are indicated to be excluded by listing them between brackets (' { }). For example: { AM } represents any amino acid other than Ala and Met. The different elements in the motif are optionally separated from each other by hyphens (-). Identical elements repeated within a motif may be indicated by placing a numerical value or a numerical range between parentheses after the element. For example, X (2) corresponds to X-X or XX; x (2, 5) corresponds to 2, 3, 4 or 5X amino acids and A (3) corresponds to A-A-A or AAA.

To distinguish between amino acids X, H and C, those amino acids in the redox motif are referred to as external amino acids X (single underlined in the above sequence) and those in the redox motif are referred to as internal amino acids X (double underlined in the above sequence).

X represents any amino acid, in particular an L-amino acid, more in particular one of the 20 naturally occurring L-amino acids.

Peptides comprising T cell epitopes and modified peptide motif sequences with reducing activity are capable of producing antigen-specific cytolytic cd4+ T cell populations directed against antigen presenting cells.

Thus, in its broadest sense, the present invention relates to a peptide comprising a T cell epitope of at least one antigen (self or non-self) having the potential to elicit an immune response, and a modified thioreductase sequence motif having reducing activity on the disulfide bond of the peptide. The T cell epitope and the modified redox motif sequence may be directly adjacent to each other in the peptide or optionally separated by one or more amino acids (so-called linker sequences). Optionally, the peptide additionally comprises an endosomal targeting sequence and/or additional "flanking" sequences.

The peptide of the present invention comprises: an MHC class II T cell epitope of an antigen (self or non-self) having the potential to elicit an immune response, and a modified redox motif. The ability of a motif sequence in a peptide to reduce sulfhydryl groups can be assayed for its reducing activity, for example in an insulin solubility assay, in which the solubility of insulin is altered upon reduction, or with a fluorescently labeled substrate such as insulin. One example of such an assay uses fluorescent peptides and is described in Tomazzolli et al (2006) Anal. Biochem.350,105-112. When two FITC-tagged peptides are covalently linked to each other through a disulfide bridge, they become self-quenching. After reduction by the peptide according to the invention, the reduced individual peptide fluoresces again.

The modified redox motif may be located on the amino-terminal side of the T cell epitope or at the carboxy-terminal end of the T cell epitope.

Peptide fragments with reducing activity are encountered in thioreductase enzymes, which are small disulfide reductases, including glutaredoxins, nucleolin toxins, thioredoxins, and other thiol/disulfide oxidoreductases (holmegren (2000) annexid. Redox signal.2,811-820;Jacquot et al (2002) biochem. Pharm.64, 1065-1069). They are multifunctional and are commonly found in many prokaryotes and eukaryotes. They exert their reducing activity on disulfide bonds on proteins (e.g. enzymes) through redox active cysteines within a conserved active domain consensus sequence: CXXC [ SEQ ID NO:18], CXXS [ SEQ ID NO:23], CXXT [ SEQ ID NO:24], SXXC [ SEQ ID NO:21], TXXXXC [ SEQ ID NO:22] (Fomenko et al (2003) Biochemistry 42,11214-11225;Fomenko et al (2002) prot.science 11, 2285-2296), wherein X represents any amino acid. Such domains are also present in larger proteins, such as Protein Disulfide Isomerase (PDI) and phosphoinositide-specific phospholipase C.

The 4 amino acid redox motif known from, for example, fomendo and WO2008/017517 comprises cysteines at positions 1 and/or 4; thus the motif is CXX [ CST ] [ SEQ ID NO:1] or [ CST ] XXC [ SEQ ID NO:2]. Such tetrapeptide sequences will be referred to as "motifs". The motif in the peptide may be any of the substitutes CXXC [ SEQ ID NO:18], SXXC [ SEQ ID NO:21], TXXXC [ SEQ ID NO:22], CXXS [ SEQ ID NO:23] or CXXT [ SEQ ID NO:24]. In particular, the peptide contains the sequence motif CXXC [ SEQ ID NO:18].

As explained in further detail, the peptides of the invention may be prepared by chemical synthesis, which allows for the incorporation of unnatural amino acids. Thus, "C" in the redox-modified redox motif described above represents cysteine or another amino acid having a thiol group, such as mercaptovaline, homocysteine or other natural or unnatural amino acid having a thiol function. In order to have reducing activity, the cysteines present in the modified redox motif should not appear as part of the cystine disulfide bridge. However, the redox-modified redox motif may comprise a modified cysteine, e.g. a methylated cysteine, which is converted in vivo to a cysteine having a free thiol group. X may be any of 20 natural amino acids, including S, C or T or may be an unnatural amino acid. In a particular embodiment, X is an amino acid having a small side chain, such as Gly, ala, ser or Thr. In a further specific embodiment, X is not an amino acid with a bulky side chain, such as Trp. In a further specific embodiment, X is not cysteine. In a further specific embodiment, at least one X in the modified redox motif is His. In other further specific embodiments, at least one X in the modified redox motif is Pro.

The peptide may further comprise modifications to increase stability or solubility, e.g. to modify the N-terminal NH ₂ A group or a C-terminal COOH group (e.g., modification of COOH to CONH ₂ A group).

In the peptides of the invention comprising a modified redox motif, the motif is positioned such that when the epitope is fitted into an MHC groove, the motif remains outside the MHC binding groove. The modified redox motif is placed next to the epitope sequence within the peptide [ in other words, the linker sequence between the motif and the epitope is zero amino acids ], or separated from the T cell epitope by a linker comprising an amino acid sequence of 5 amino acids or less. More particularly, the linker comprises 1, 2, 3, 4 or 5 amino acids. Particular embodiments are peptides having a 0, 1 or 2 amino acid linker between the epitope sequence and the modified redox motif sequence. In those peptides where the modified redox motif sequence is adjacent to the epitope sequence, this is indicated as positions P-4 to P-1 or P+1 to P+4 compared to the epitope sequence. In addition to peptide linkers, other organic compounds may also be used as linkers to link portions of the peptide to each other (e.g., linking modified redox motif sequences to T cell epitope sequences).

The peptides of the invention may further comprise additional short amino acid sequences at the N-or C-terminus of the sequence comprising the T cell epitope and the modified redox motif. Such amino acid sequences are generally referred to herein as 'flanking sequences'. Flanking sequences may be located between the epitope and the endosomal targeting sequence and/or between the modified redox motif and the endosomal targeting sequence. In certain peptides that do not include an endosomal targeting sequence, a short amino acid sequence may be present at the N and/or C terminus of the modified redox motif and/or epitope sequence within the peptide. More particularly, the flanking sequences are sequences of 1 to 7 amino acids, most particularly 2 amino acids.

The modified redox motif may be located at the N-terminus of the epitope.

In certain embodiments of the invention, peptides comprising one epitope sequence and a modified redox motif sequence are provided. In further specific embodiments, the modified redox motif occurs multiple times (1, 2, 3, 4 or even more times) in the peptide, for example as repeats of the modified redox motif that may be spaced from one another by one or more amino acids, or as repeats immediately adjacent to one another. Alternatively, one or more modified redox motifs are provided at both the N-and C-terminus of the T cell epitope sequence.

Other variants contemplated for the peptides of the invention include peptides comprising repeats of T cell epitope sequences, wherein each epitope sequence is preceded and/or followed by a modified redox motif (e.g., a repeat of a "modified redox motif-epitope" or a repeat of a "modified redox motif-epitope-modified redox motif"). In this context, the modified redox motifs may all have the same sequence, but this is not mandatory. Notably, a repeat sequence comprising a peptide that itself contains an epitope of a modified redox motif will also result in a sequence comprising an 'epitope' and a 'modified redox motif'. In these peptides, the modified redox motif within one epitope sequence acts as a modified redox motif outside the second epitope sequence.

Typically, the peptides of the invention comprise only one T cell epitope. T cell epitopes in the protein sequence may be identified by one or more of a functional assay and/or a silica prediction assay, as described below. Amino acids in the T cell epitope sequence are numbered according to their position in the MHC protein binding groove. The T cell epitope present within the peptide consists of 8 to 25 amino acids, more particularly 8 to 16 amino acids, but most particularly 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.

In a more specific embodiment, the T cell epitope consists of a 9 amino acid sequence. In a further specific embodiment, the T cell epitope is an epitope presented to the T cell by an MHC-II molecule [ MHC class II restricted T cell epitope ]. Typically, a T cell epitope sequence refers to an octapeptide or more specifically a nonapeptide sequence suitable for cleavage of MHC II proteins.

The T cell epitope of the peptide of the invention may correspond to the native epitope sequence of the protein or may be a modified version thereof, provided that the modified T cell epitope retains its ability to bind within an MHC cleft, similar to the native T cell epitope sequence. The modified T cell epitope may have the same binding affinity for MHC proteins as the native epitope, but may also have a reduced affinity. In particular, the binding affinity of the modified peptide is not less than 10-fold, more particularly not less than 5-fold lower than that of the original peptide. The peptides of the invention have a stabilizing effect on protein complexes. Thus, stabilization of the peptide-MHC complex compensates for the reduced affinity of the modified epitope for the MHC molecule.

The sequence comprising the T cell epitope and the reducing compound within the peptide may be further linked to an amino acid sequence (or another organic compound) that facilitates uptake of the peptide into late endosomes for processing and presentation within MHC class II determinants. Late endosomal targeting is mediated by signals present in the cytoplasmic tail of proteins and corresponds to well-identified peptide motifs. Late endosomal targeting sequences allow MHC class II molecules to process and efficiently present antigen-derived T cell epitopes. Such endosomal targeting sequences are contained, for example, in gp75 protein (Vijayasaradi et al (1995) J.cell. Biol.130, 807-820), human CD3 gamma protein, HLA-BM 11 (Copier et al (1996) J.Immunol.157, 1017-1027), cytoplasmic tail of the DEC205 receptor (Mahnke et al (2000) J.cell biol.151, 673-683). Other examples of peptides that exert sorting signals to endosomes are disclosed in the reviews of Bonifacio and Traub (2003) annu.rev. Biochem.72, 395-447. Alternatively, the sequence may be a sequence from a subdominant or minor T cell epitope of the protein that facilitates uptake in late endosomes without overcoming a T cell response to the antigen. Late endosomal targeting sequences may be located at the amino-or carboxy-terminus of the antigen-derived peptide for efficient uptake and processing, and may also be coupled by flanking sequences such as peptide sequences of up to 10 amino acids. When a minor T cell epitope is used for targeting purposes, the latter is typically located at the amino terminus of the antigen-derived peptide.

Thus, the present invention contemplates peptides of antigenic proteins and their use in eliciting specific immune responses. These peptides may correspond to protein fragments comprising within their sequence a T cell epitope and a reducing compound separated by up to 10, preferably 7 or less amino acids. Alternatively, and for most antigenic proteins, the peptides of the invention are produced by coupling a reducing compound, more particularly a reduction modified redox motif as described herein, at the N-or C-terminus to a T cell epitope of the antigenic protein (either directly adjacent thereto or having a linker of up to 10, more particularly up to 7 amino acids). In addition, the T cell epitope sequence and/or modified redox motif of the protein may be modified and/or one or more flanking sequences and/or targeting sequences may be introduced (or modified) as compared to the naturally-occurring sequence. Thus, depending on whether the features of the invention can be found within the sequence of the antigen protein of interest, the peptides of the invention may comprise a sequence that is 'artificial' or 'naturally occurring'.

The length of the peptides of the invention can vary significantly. Peptides may vary in length from 13 or 14 amino acids (i.e., consisting of an epitope of 8-9 amino acids, 5 amino acids of the modified redox motif adjacent thereto with histidine) up to 20, 25, 30, 40 or 50 amino acids. For example, the peptide may comprise an endosomal targeting sequence of 40 amino acids, a flanking sequence of about 2 amino acids, a 5 amino acid motif as described herein, a 4 amino acid linker, and a 9 amino acid T cell epitope peptide.

Thus, in particular embodiments, the intact peptide consists of 13 amino acids up to 20, 25, 30, 40, 50, 75 or 100 amino acids. More particularly, where the reducing compound is a modified redox motif as described herein, the length of the (artificial or natural) sequence comprising the epitope optionally linked by a linker and the modified redox motif (referred to herein as the 'epitope-modified redox motif' sequence) but not comprising the endosomal targeting sequence is critical. The 'epitope-modified redox motif' more particularly has a length of 13, 14, 15, 16, 17, 18 or 19 amino acids. Such 13 or 14 to 19 amino acid peptides may optionally be coupled to an endosomal targeting signal, the size of which is less important.

As described above, in certain embodiments, the peptides of the invention comprise a redox motif as described herein that is reduction modified in connection with a T cell epitope sequence.

In a further specific embodiment, the peptide of the invention is a peptide comprising a T cell epitope that does not comprise within its native sequence an amino acid sequence having redox properties.

However, in alternative embodiments, a T cell epitope may comprise any amino acid sequence that ensures binding of the epitope to an MHC cleft. When the target epitope of the antigenic protein comprises within its epitope sequence a modified redox motif as described herein, the immunogenic peptide according to the invention comprises a modified redox motif sequence as described herein and/or a sequence of another reduction sequence coupled to the N-or C-terminus of the epitope sequence such that (as opposed to the modified redox motif present within the epitope embedded within the cleft) the attached modified redox motif can ensure the reducing activity.

Thus, T cell epitopes and motifs are directly adjacent or separate from each other, but do not overlap. To evaluate the concept of "direct adjacent" or "split", 8 or 9 amino acid sequences suitable for within an MHC cleft were determined, and the distance between such octapeptide or nonapeptide and the redox motif tetrapeptide or modified redox motif pentapeptide (including histidine) was determined.

Typically, the peptides of the invention are not native (and thus are not as such protein fragments), but are artificial peptides which in addition to a T cell epitope contain a modified redox motif as described herein, whereby the modified redox motif is directly separated from the T cell epitope by a linker consisting of up to seven, most particularly up to four or up to 2 amino acids.

It has been shown that when a peptide according to the invention (or a composition comprising such a peptide) is administered (i.e. injected) to a mammal, the peptide triggers activation of T cells, recognizes antigen-derived T cell epitopes and provides additional signals to T cells through reduction of surface receptors. This super-optimal activation results in T cells obtaining cytolytic properties against T cell epitope presenting cells, as well as suppressive properties against bystander T cells. In this way, the peptides of the invention or compositions comprising the peptides containing antigen-derived T cell epitopes and modified redox motifs outside of the epitopes can be used for direct immunization of mammals, including humans. Accordingly, the present invention provides a peptide of the invention or a derivative thereof for use as a medicament. Accordingly, the present invention provides a method of treatment comprising administering one or more peptides according to the invention to a patient in need thereof.

The present invention provides a method by which antigen-specific T cells with cytolytic properties can be primed by immunization with small peptides. It has been found that peptides containing (i) sequences encoding T cell epitopes from antigens and (II) consensus sequences with redox properties and further optionally also sequences promoting peptide uptake into late endosomes for efficient MHC class II presentation, elicit suppressor T cells.

The immunogenic properties of the peptides of the invention are of particular interest in the treatment and prevention of immune responses.

The peptides described herein are useful as medicaments, more particularly for the manufacture of medicaments for the prevention or treatment of immune disorders in mammals, particularly humans.

The present invention describes a method for treating or preventing an immune disorder in a mammal in need of such treatment or prevention by using a peptide of the invention, a homologue or derivative thereof, the method comprising the steps of: administering to said mammal suffering from or at risk of an immune disorder a therapeutically effective amount of a peptide of the invention, a homologue or derivative thereof, thereby alleviating the symptoms of an immune disorder. Treatment of humans and animals such as pets and farm animals is contemplated. In one embodiment, the mammal to be treated is a human. The immune disorder described above is selected in particular embodiments from allergic diseases and autoimmune diseases.

The peptides of the invention or pharmaceutical compositions comprising such peptides as defined herein are preferably administered subcutaneously or intramuscularly. Preferably, the peptide or pharmaceutical composition comprising such peptide may be injected Subcutaneously (SC) in the region of the outer portion of the upper arm intermediate between the elbow and shoulder. When two or more separate injections are required, they may be administered simultaneously in both arms.

The peptide according to the invention or the pharmaceutical composition comprising the same is administered in a therapeutically effective dose. An exemplary but non-limiting dosage regimen is 50 to 1500 μg, preferably 100 to 1200 μg. More specific dosage regimens may be between 50 and 250 μg, between 250 and 450 μg, or between 850 and 1300 μg, depending on the condition of the patient and the severity of the disease. The dosage regimen may comprise administration of a single dose or simultaneous or sequential administration of 2, 3, 4, 5 or more doses. An exemplary, non-limiting dosing regimen is as follows:

a low dose regimen comprising SC administration of 50 μg of peptide, two injections of 25 μg each (100 μl each), followed by three consecutive injections of 25 μg of peptide, two injections of 12.5 μg each (50 μl each).

Medium dose regimen comprising SC administration of 150 μg of peptide, in two injections of 75 μg each (300 μl each), followed by three consecutive administrations of 75 μg of peptide, in two injections of 37.5 μg each (150 μl each).

High dose regimen comprising SC administration of 450 μg of peptide, in two injections of 225 μg each (900 μl each), followed by three consecutive administrations of 225 μg of peptide, in two injections of 112.5 μg each (450 μl each).

For all the above peptides other variants are envisaged, wherein one or two amino acids X are present between histidine and cysteine. Typically, these external amino acids X are not His, cys, ser or Thr.

The peptides of the invention can also be used in vitro diagnostic methods for detecting class II restricted cd4+ T cells in a sample. In this method, the sample is contacted with a complex of an MHC class II molecule and a peptide according to the invention. Cd4+ T cells are detected by measuring the binding of the complex to cells in the sample, wherein binding of the complex to cells is indicative of the presence of cd4+ T cells in the sample.

The complex may be a fusion protein of a peptide and an MHC class II molecule.

Alternatively, the MHC molecules in the complex are tetramers. The complex may be provided as a soluble molecule or may be attached to a carrier.

Thus, in particular embodiments, the therapeutic and prophylactic methods of the invention comprise administering an immunogenic peptide as described herein, wherein the peptide comprises a T cell epitope of an antigenic protein that plays a role in the disease to be treated (e.g., those described above). In a further specific embodiment, the epitope used is a dominant epitope.

The peptides according to the invention are prepared by synthesizing peptides in which the T cell epitope and the modified redox motif are separated by 0 to 5 amino acids. In certain embodiments, modified redox motifs can be obtained by introducing 1, 2 or 3 mutations outside the epitope sequence to preserve the sequence context present in the protein. Generally, with reference to the nonapeptide as part of the native sequence, the amino acids in P-2 and P-l and P+10 and P+11 remain in the peptide sequence. These flanking residues generally stabilize binding to MHC class II. In other embodiments, the sequence N-terminus or C-terminus of the epitope is independent of the sequence of the antigen protein containing the T cell epitope sequence.

Thus, peptides are produced by chemical peptide synthesis, recombinant expression methods or, in more specific cases, proteolytic or chemical fragmentation of proteins, based on the above-described methods of designing peptides.

Peptides produced in the above methods can be tested for the presence of T cell epitopes in vitro and in vivo methods, and their reductive activity can be tested in vitro assays. As a final quality control, these peptides can be tested in an in vitro assay to verify whether the peptides can produce cd4+ T cells that lyse antigen presenting cells via the apoptotic pathway that present antigens containing epitope sequences that are also present in peptides with modified redox motifs.

The peptides of the invention can be produced in bacteria, yeast, insect cells, plant cells, or mammalian cells using recombinant DNA techniques. Given the limited length of peptides, they can be prepared by chemical peptide synthesis, in which peptides are prepared by coupling different amino acids to each other. Chemical synthesis is particularly suitable for incorporating, for example, D-amino acids, amino acids with non-naturally occurring side chains or natural amino acids with modified side chains, such as methylated cysteines.

Chemical peptide synthesis methods are well described and peptides can be ordered from companies such as Applied Biosystems and others.

Peptide synthesis may be performed as Solid Phase Peptide Synthesis (SPPS) or as opposed to solution phase peptide synthesis. The most notable SPPS methods are t-Boc and Fmoc solid phase chemistry:

during peptide synthesis, a variety of protecting groups are used. For example, hydroxy and carboxy functional groups are protected by t-butyl, lysine and tryptophan are protected by t-Boc groups, asparagine, glutamine, cysteine and histidine are protected by trityl groups, and arginine is protected by pdf groups. Such protecting groups may be left on the peptide after synthesis, if appropriate. Using a ligation strategy (chemoselective coupling of two unprotected peptide fragments), peptides can be ligated to each other to form longer peptides, as originally described by Kent (Schnelzer & Kent (1992) int. J. Pept. Protein Res.40, 180-193) and reviewed, for example, by Tam et al (2001) Biopolymers 60,194-205, which offers great potential beyond SPPS to achieve protein synthesis. Many proteins of 100-300 residues in size have been successfully synthesized by this method. Due to the tremendous progress of SPPS, synthetic peptides continue to play an increasingly important role in the research fields of biochemistry, pharmacology, neurobiology, enzymology, molecular biology, and the like.

Alternatively, the peptide may be synthesized in a suitable expression vector comprising the coding nucleotide sequence by using a nucleic acid molecule encoding the peptide of the invention. Such DNA molecules can be readily prepared using an automated DNA synthesizer and the well known codon-amino acid relationships of the genetic code. Such DNA molecules may also be obtained as genomic DNA or cDNA using oligonucleotide probes and conventional hybridization methods. Such DNA molecules may be incorporated into expression vectors (including plasmids) suitable for expressing DNA and producing polypeptides in a suitable host such as bacteria (e.g., e.coli), yeast cells, animal cells, or plant cells.

Physical and chemical properties (e.g., solubility, stability) of the peptide of interest are examined to determine whether the peptide is suitable for use in a therapeutic composition. Typically, this is optimized by modulating the sequence of the peptide. Optionally, the peptides may be modified after synthesis using techniques well known in the art (chemical modifications, e.g., addition/deletion of functional groups).

T cell epitopes are thought to themselves trigger early events in T helper cell levels by binding to appropriate HLA molecules on the surface of antigen presenting cells and stimulating relevant T cell subsets. These events lead to T cell proliferation, lymphokine secretion, local inflammatory response, recruitment of additional immune cells to the site, and activation of B cell cascades leading to antibody production. One isotype of these antibodies lgE is of fundamental importance in the development of allergic symptoms and its production is affected at the T helper cell level by the nature of secreted lymphokines early in the event cascade. T cell epitopes are the basic element or minimal unit of T cell receptor recognition, wherein an epitope comprises amino acid residues necessary for receptor recognition that are contiguous in the amino acid sequence of a protein.

However, upon administration of peptides having T cell epitopes and redox motifs, the following events are believed to occur:

activation of antigen (i) specific T cells by homologous interaction with antigen-derived peptides presented by MHC class II molecules;

the reductase sequence reduces T cell surface proteins, such as CD4 molecules, whose second domain contains a restricted disulfide bridge. This transduces the signal to T cells. Among the series of consequences associated with increased oxidative pathways are increased calcium influx and translocation of NF-kB transcription factors to the nucleus. The latter results in increased transcription of IFN-gamma and granzymes, such that the cells acquire cytolytic properties by inducing apoptotic mechanisms; the cytolytic properties affect peptide-presenting cells by mechanisms involving granzyme B secretion and Fas-FasL interactions. Since cell killing is achieved through apoptotic pathways, cytolytic cells are a more appropriate term for these cells than cytotoxic cells. Disruption of antigen presenting target cells prevents activation of other T cells specific for epitopes located on the same antigen, or activation of unrelated antigens processed by the same antigen presenting cells; another consequence of T cell activation is the inhibition of bystander T cell activation by cell-cell contact dependent mechanisms. In this case, if cytolytic T cells and bystander T cells are in close proximity, i.e. activated on the surface of the same antigen presenting cell, then antigen-activated T cells presented by different antigen presenting cells are also inhibited.

The postulated mechanism of action is demonstrated by experimental data disclosed in the above-cited PCT application WO2008/017517 and the present inventors publications.

The present invention provides methods for producing antigen-specific cytolytic cd4+ T cells in vivo or in vitro, and methods for distinguishing cytolytic cd4+ T cells from other cell populations (e.g., foxp3+ Tregs) based on characteristic expression data, independent of such methods.

The present invention describes in vivo methods for generating antigen specific cd4+ T cells. One particular embodiment relates to a method for producing or isolating cd4+ T cells by immunizing an animal (including a human) with a peptide of the invention described herein, and then isolating the cd4+ T cells from the immunized animal. The present invention describes in vitro methods for generating antigen specific cytolytic cd4+ T cells against APC. The present invention provides methods of producing antigen-specific cytolytic CD4+ T cells directed against APCs.

In one embodiment, there is provided a method comprising: peripheral blood cells are isolated, the cell population is stimulated in vitro by the immunogenic peptides according to the invention, and the stimulated cell population is expanded, in particular in the presence of IL-2. The method according to the present invention has the advantage of producing a large number of cd4+ T cells and can produce cd4+ T cells specific for antigen proteins (by using peptides comprising antigen-specific epitopes).

In alternative embodiments, cd4+ T cells may be produced in vivo, i.e., by injecting an immunogenic peptide as described herein into a subject, and collecting the cytolytic cd4+ T cells produced in vivo.

Antigen specific cytolytic cd4+ T cells against APCs obtainable by the method of the invention are particularly useful for administration to mammals for immunotherapy, prevention of allergic reactions and treatment of autoimmune diseases. The use of allogeneic and autologous cells is contemplated.

Cytolytic cd4+ T cell populations were obtained as described below.

Antigen-specific cytolytic cd4+ T cells as described herein are useful as medicaments, more particularly for adoptive cell therapy, more particularly for treating acute allergic reactions and the recurrence of autoimmune diseases (such as multiple sclerosis). An isolated cytolytic cd4+ T cell or cell population, more particularly an antigen specific cytolytic cd4+ T cell population, for use in the manufacture of a medicament for the prevention or treatment of an immune disorder. Methods of treatment by use of isolated or generated cytolytic CD4+ T cells are disclosed.

As explained in WO2008/017517, cytolytic cd4+ T cells against APC can be distinguished from natural Treg cells based on the expression characteristics of the cells. More specifically, the cytolytic cd4+ T cell population exhibits one or more of the following characteristics compared to the natural population of Treg cells:

Increased expression of surface markers including CD103, CTLA-4, fasl and ICOS upon activation;

intermediate expression of CD25;

expresses CD4, ICOS, CTLA-4, GITR, and either low or non-expressing CD127 (IL 7-R), non-expressing CD27;

the transcription factors T-bet and egr-2 (Krox-20) are expressed, but the transcription repressor Foxp3 is not expressed;

IFN-gamma is produced in high yield and IL-10, IL-4, IL-5, IL-13 or TGF-beta is not produced or is produced only in trace amounts.

In addition, cytolytic T cells express CD45RO and/or CD45RA, do not express CCR7, CD27 and present high levels of granzyme B and other granzymes as well as Fas ligand.

The peptides of the invention, when administered to living animals (typically humans), elicit specific T cells that will exert inhibitory activity against bystander T cells.

In a specific embodiment, the cytolytic cell population of the invention is characterized by expression of FasL and/or interferon gamma. In a specific embodiment, the cytolytic cell population of the invention is further characterized by expression of granzyme B.

The mechanism also suggests and experimental results show that the peptides of the invention, although comprising specific T cell epitopes of a certain antigen, can be used for preventing or treating disorders caused by immune responses to other T cell epitopes of the same antigen, or in some cases even for treating disorders caused by immune responses to other T cell epitopes of other different antigens, if the other different antigens would be presented via the same mechanism by MHC class II molecules in the vicinity of T cells activated by the peptides of the invention.

Isolated cell populations of cell types having the above characteristics are disclosed that, in addition to being antigen specific, are capable of suppressing antigen specific immune responses.

The present invention provides a pharmaceutical composition comprising one or more peptides according to the invention, further comprising a pharmaceutically acceptable carrier. As mentioned above, the invention also relates to a composition for use as a medicament or to a method of treating an immune disorder in a mammal by using said composition and to the use of the composition for the manufacture of a medicament for the prevention or treatment of an immune disorder. For example, the pharmaceutical composition may be a vaccine suitable for the treatment or prevention of immune disorders (in particular airborne and food-borne allergies) and allergic diseases. As an example of a pharmaceutical composition as further described herein, a peptide according to the invention is adsorbed onto an adjuvant suitable for administration to a mammal, such as aluminium hydroxide (alum). Typically, 50 μg of peptide adsorbed on alum was injected 3 times via subcutaneous route, 2 weeks apart. It will be apparent to those skilled in the art that other routes of administration are possible, including oral, intranasal, or intramuscular. Moreover, the number of injections and the amount of injections may vary depending on the condition to be treated. In addition, adjuvants other than alum can be used provided that they promote MHC class II presentation and peptide presentation in T cell activation. Thus, although the active ingredients may be administered alone, they are typically provided as pharmaceutical formulations. The veterinary and human formulations of the present invention comprise at least one active ingredient as described above together with one or more pharmaceutically acceptable carriers. The present invention relates to pharmaceutical compositions comprising one or more peptides according to the invention as active ingredient in admixture with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention should contain a therapeutically effective amount of the active ingredient, as shown below with respect to the therapeutic or prophylactic method. Optionally, the composition further comprises other therapeutic ingredients. Suitable other therapeutic ingredients, as well as their usual dosages, are well known to those skilled in the art, depending on the class to which they belong, and may be selected from other known drugs for the treatment of immune disorders.

The term "pharmaceutically acceptable carrier" as used herein refers to any material or substance formulated with an active ingredient to facilitate application or transmission of the active ingredient to the site to be treated, for example by dissolving, dispersing or diffusing the composition, and/or to assist in its storage, transport or handling without compromising its effectiveness. They include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents (e.g., phenol, sorbic acid, chlorobutanol), isotonic agents (e.g., sugars or sodium chloride), and the like. Additional ingredients may be included to control the duration of action of the immunogenic peptide in the composition. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the composition of the invention may suitably be used as a concentrate, emulsion, solution, granule, powder, spray, aerosol, suspension, ointment, cream, tablet, pill or powder. Suitable pharmaceutical carriers for pharmaceutical compositions and formulations thereof are well known to those skilled in the art and their choice is not particularly limited in the present invention. They may also include additives such as wetting agents, dispersing agents, sticking agents, binders, emulsifiers, solvents, coatings, antibacterial and antifungal agents (e.g., phenol, sorbic acid, chlorobutanol), isotonic agents (e.g., sugars or sodium chloride), and the like, provided that they are compatible with pharmaceutical practices, i.e., carriers and additives that do not cause permanent damage to mammals. The pharmaceutical compositions of the invention may be prepared in any known manner, for example by homogeneously mixing, coating and/or grinding the active ingredient together with the selected carrier material and, where appropriate, further additives such as surfactants, in a one-step or multi-step procedure. They can also be prepared by micronization, for example to obtain the form of microspheres, generally having a diameter of about 1 to 10 μm, i.e. for the manufacture of microcapsules for controlled or sustained release of the active ingredient.

Suitable surfactants to be used in the pharmaceutical compositions of the present invention, also known as leakage-promoting agents or emulsifiers, are nonionic, cationic and/or anionic materials having good emulsifying, dispersing and/or wetting properties. Suitable anionic surfactants include water-soluble soaps and water-soluble synthetic surfactants. Suitable soaps are the alkali metal or alkaline earth metal salts of higher fatty acids (C10-C22), the unsubstituted or substituted ammonium salts, for example the sodium or potassium salts of oleic or stearic acid, or the sodium or potassium salts of natural fatty acid mixtures obtainable from coconut oil or tallow. Synthetic surfactants include sodium or calcium salts of polyacrylic acid; fatty sulfonates and sulfates; sulfonated benzimidazole derivatives and alkylaryl sulfonates. Fatty sulphonates or sulphates are generally in the form of alkali metal or alkaline earth metal salts, unsubstituted ammonium salts or ammonium salts substituted with alkyl or acyl groups having from 8 to 22 carbon atoms, for example sodium or calcium salts of lignin sulphonic acid or dodecyl sulphonic acid or mixtures of fatty alcohol sulphates obtained from natural fatty acids, alkali metal or alkaline earth metal salts of sulphuric acid or sulphonates (such as sodium dodecyl sulphate) and alkali metal or alkaline earth metal salts of sulphonic acids of fatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole derivatives generally contain from 8 to 22 carbon atoms. Examples of alkylaryl sulfonates are sodium, calcium or alkanolamine salts of dodecylbenzenesulfonic acid or dibutylnaphthalenesulfonic acid or naphthalenesulfonic acid/formaldehyde condensation products (alcanolamine salt). Also suitable are the corresponding phosphates, for example salts of phosphoric acid esters, and adducts of p-nonylphenol with ethylene oxide and/or propylene oxide, or phospholipids. Suitable phospholipids for this purpose are natural (derived from animal or plant cells) or synthetic cephalins or lecithins such as, for example, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, lysolecithin, cardiolipin, dioctyl phosphatidylcholine, dipalmitoyl phosphatidylcholine and mixtures thereof.

Suitable nonionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides having at least 12 carbon atoms in the molecule, alkylaromatic sulfonates and dialkylsulfosuccinates, such as polyethylene glycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives typically having 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Other suitable nonionic surfactants are water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylene diamino-polypropylene glycol having 1 to 10 carbon atoms in the alkyl chain, containing 20 to 250 glycol ether groups and/or 10 to 100 propylene glycol ether groups. These compounds generally contain 1 to 5 ethylene glycol units per propylene glycol unit. Representative examples of nonionic surfactants are nonylphenol-polyethoxyethanol, castor oil polyglycol ether, polypropylene/polyethylene oxide adducts, tributylphenoxy polyethoxyethanol, polyethylene glycol, and octylphenoxy polyethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable nonionic surfactants. Suitable cationic surfactants include quaternary ammonium salts having 4 hydrocarbyl groups optionally substituted with halogen, phenyl, substituted phenyl or hydroxy groups, particularly halides; for example, quaternary ammonium salts containing at least one C8C22 alkyl group (e.g., cetyl, lauryl, palmityl, myristyl, oleyl, etc.) as an N-substituent and containing unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy-lower alkyl groups as further substituents.

A more detailed description of surfactants suitable for this purpose can be found, for example, in "McCutcheon's annual journal of detergents and emulsifiers (McCutcheon's Detergents and Emulsifiers Annual)" (MC Publishing crop, ridgewood, new Jersey, 1981), "Tensid-Taschenbucw ',2 d. (Hanser Verlag, vienna, 1981) and" encyclopedia of surfactants (Encyclopaedia of Surfactants), (Chemical Publishing Co., new York, 1981). The peptides according to the invention, their homologues or derivatives (and their physiologically acceptable salts or pharmaceutical compositions, all included in the term "active ingredient") may be administered by any route suitable for the condition to be treated and for the compound (here proteins and fragments) to be administered. Possible routes include regional, systemic, oral (solid form or inhaled), rectal, nasal, topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraarterial, intrathecal and epidural). The preferred route of administration may vary depending upon, for example, the condition of the recipient or the condition to be treated. As described herein, the carrier is optimally "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraarterial, intrathecal and epidural) administration.

Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous or nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Typical unit dose formulations are those containing a daily dose or unit daily sub-dose (as described above or an appropriate fraction thereof) of the active ingredient. It should be understood that the formulations of the present invention may include other agents conventional in the art with respect to the type of formulation in question, in addition to the ingredients specifically mentioned above, for example, those suitable for oral administration may include flavoring agents. The peptides, homologues or derivatives thereof according to the present invention may be used to provide controlled release pharmaceutical formulations ("controlled release formulations") containing as active ingredient one or more compounds of the present invention, wherein the release of the active ingredient may be controlled and regulated to allow for less frequent administration or to improve the pharmacokinetic or toxicity profile of a given compound of the present invention. Controlled release formulations suitable for oral administration may be prepared according to conventional methods, wherein the discrete units comprise one or more compounds of the invention. Additional ingredients may be included to control the duration of action of the active ingredients in the composition. Thus, controlled release compositions can be achieved by selecting suitable polymeric carriers such as polyesters, polyamino acids, polyvinylpyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like. The rate of drug release and duration of action can also be controlled by incorporating the active ingredient into particles (e.g., microcapsules) of polymeric materials such as hydrogels, polylactic acid, hydroxymethyl cellulose, polymethyl methacrylate and other such polymers. These include colloidal drug delivery systems such as liposomes, microspheres, microemulsions, nanoparticles, nanocapsules, and the like. Depending on the route of administration, the pharmaceutical composition may require a protective coating. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation thereof. Typical carriers for this purpose therefore include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol and the like and mixtures thereof. Whereas when multiple active ingredients are used in combination they do not necessarily exert their combined therapeutic effect directly in the mammal to be treated at the same time, the corresponding compositions may also be in the form of a pharmaceutical kit or package, wherein the two ingredients are contained in separate but adjacent reservoirs or compartments. In the latter case, each active ingredient may thus be formulated in a manner suitable for a route of administration different from that of the other ingredients, for example one of which may be in the form of an oral or parenteral formulation, and the other may be in the form of an ampoule or aerosol for intravenous injection.

As demonstrated in vitro and in vivo, cytolytic cd4+ T cells obtained in the present invention induce APC apoptosis following MHC-II dependent cognate activation, affecting dendritic cells and B cells, and (2) inhibit bystander T cells by contact-dependent mechanisms in the absence of IL-10 and/or TGF- β. As discussed in detail in WO2008/017517, cytolytic cd4+ T cells may be distinguished from natural and adaptive tregs.

The invention will now be illustrated by the following examples, which are provided without any limiting intent. Further, all references described herein are expressly incorporated herein by reference.

Examples

Example 1: peptide design

In contrast to the peptides disclosed in WO2016059236, peptides comprising T cell epitopes of the C domain of insulin were synthesized in which VR dipeptide sequences not present in the insulin sequence have been removed, as shown in the alignment described below:

P17 001：HCPYC VR SLQPLALEGSLQKRG[SEQ ID NO:25]

P17 003：HCPYC—SLQPLALEGSLQKRG[SEQ ID NO:26]

thus, the P17 003 peptide contains a CxC [ SEQ ID NO:18] motif preceded by His, where xx is Pro and Tyr. The 9 amino acid T cell epitope has the sequence LALEGSLQK [ SEQ ID NO:3] and is separated from the CxC [ SEQ ID NO:18] motif by a 4 amino acid SLQP linker. RG dipeptide is the c-terminal flanking sequence of the epitope. In this peptide, sequence SLQPLALEGSLQKRG [ SEQ ID NO:20] has 100% identity to the sequence that occurs in insulin.

Example 2: method for evaluating peptide reduction activity

The reductase activity of the peptides was assayed using fluorescence as described in tomazzoli et al (2006) al biochem.350, 105-112. Two peptides with FITC labels become self-quenching when covalently linked to each other through a disulfide bridge. After reduction by the peptide according to the invention, the reduced individual peptide again becomes fluorescent.

Control experiments were performed with dithiothreitol (100% reduction activity) and water (100% reduction activity).

Peptide P17 001 showed 68% of the reducing activity, while peptide P17 003 showed 65% of the reducing activity.

Example 3: interferon gamma release from cytolytic CD4+ T cell lines

Interferon gamma is an important marker for characterizing cytolytic CD4+ T cells.

Specific cd4+ T cell lines were obtained by priming and stimulating naive cd4+ T cells from T1D patients (T1D 07) with the peptide p 17-001. After 12 stimulations, cells were co-cultured with autologous LCL B cells loaded with (2. Mu.M) peptide p17-001 or p 17-003. After 24 hours, the supernatant was collected and IFN-gamma was measured by multiplex assay (see Table 1 below).

TABLE 1

	Stimulus 1
			IFN-γ(pg/ml)
p17 UL	2.35±1.2
		p17 001	11.1±0.8
p17 003	29.7±14.1

There was a significant difference in IFN-gamma production between the two peptides (approximately 3-fold more IFN-gamma was produced after stimulation with p17 003 compared to p17 001).

Example 4: fasL release from cytolytic CD4+ T cell lines

The T cell line originally generated with P17001 as described in example 3 above was split and stimulated with peptide P17003 or P17001 in 4 consecutive in vitro stimulations using the autologous LCL B cell line as APC. On day 11 of each stimulation (4 total), cells were tested for FasL after restimulation with the corresponding peptide presented by autologous B cells. Supernatants were collected after 24 hours (stimulation 1 and 2) or 72 hours (stimulation 3 and 4) of co-culture.

TABLE 2 FasL expression of CD4+ T cell lines from T1D patients

For each of the four stimuli, fasL (also known as sFasL) expression of P17-003 was significantly higher.

This suggests that the P17-003 peptide is more capable of producing cytolytic T cells than the P17-001 peptide.

Example 5: release of sFasL and cytokine production in PBMC of T1D patients.

PBMCs from T1D018 patients with T1D were stimulated in vitro with P17001 peptide or P17003 peptide. Both populations specifically released Il-5 upon antigen activation. After 6 stimulation cycles with peptide, cytokine capture beads were used to enrich both cell lines for IL-5-producing cells. Two populations of interleukin-5 negative cells were used as controls.

The four groups were then tested for specific release of sFasL, granzyme B and cytokines after stimulation with their cognate peptides (P17-001 or P17-003). After 24 hours of incubation, the supernatant was collected, sFasL and granzyme B (sFasL: diacetone 851730010; granzyme B: eBioscience BMS 2027) were measured by ELISA and cytokines were measured by MACSPLex cytokine 12 kit (Miltenyi, 130-099-169).

sFasL production

sFasL levels for the four cell lines are shown in figure 1. This shows that the IL5 positive fraction (black histogram) enriched for specific cells specific for peptide p17-003 or p17-001 released more sFasL than the negative fraction (open histogram), indicating efficient specific cell purification.

Furthermore, the results show that cell-specific release by stimulation in vitro with p17-003 is significantly 4.5-fold more sFasL (p < 0.0001) compared to cells produced by peptide p 17-001.

5.2. Granzyme B production

The IL5 positive fraction of cells enriched for specific cells specific for the peptide p17-003 or p17-001 was tested, releasing more granzyme B than the IL5 negative fraction, which was used as a measure of effective specific cell purification.

In addition, the release of granzyme B, which produced an increased specificity of cells stimulated in vitro with p17-003, will be tested compared to cells produced by the peptide p 17-001.

5.3. Cytokine release

To determine whether cultured cells from T1D donors were specific for peptide P17001 or P17003, cytokine release was studied using MACSplex cytokine 12 kit (Miltenyi, 130-099-169), which is a marker of cell activation following peptide stimulation. After incubation of T1D donor PBMCs for 24 hours in the absence or presence of peptide, the supernatant was collected. Cytokine concentrations of biological replicates were determined and shown in pg/mL. Specificity tests were performed at the end of stimulus 10 (holiday). The results are expressed as the concentration differences for each cytokine in the absence and presence of peptide.

FIG. 2 shows that the IL5 positive part of the P17003 cell line responds specifically to stimuli compared to the cell lines P17003 IL5 negative, P17001 IL5 negative and P17001 IL5 positive. This suggests that peptide P17003 is more efficient at eliciting peptide-specific cells than P17001.

In summary, P17-003 regenerated cells were more efficient at releasing lytic molecules (such as sFasL and latent granzyme B) and cytokines than cells produced with P17-001. Thus, the peptides result in cd4+ cell populations having excellent cytolytic properties for APCs presenting insulin epitopes.

Example 6: clinical trial

Prior to administration, the peptides of the invention are reconstituted with a diluent containing an adjuvant. The product should be reconstituted temporarily, preferably for use less than 3 hours after reconstitution.

The study drug product may be formulated such that the concentration of peptide in the vial is 250 μg/ml after reconstitution in the diluent. An appropriate volume will be removed to conform to the clinical trial protocol. For example:

the low dose (group 1) may comprise SC administration of 50 μg of peptide, two injections of 25 μg each (100 μl each), followed by three consecutive injections of 25 μg of peptide, two injections of 12.5 μg each (50 μl each).

The medium dose (group 2) may comprise SC administration of 150 μg of peptide, in two injections of 75 μg each (300 μl each), followed by three consecutive administrations of 75 μg of peptide, in two injections of 37.5 μg each (150 μl each).

High dose (group 3) may comprise SC administration of 450 μg of peptide, in two injections of 225 μg each (900 μl each), followed by three consecutive administrations of 225 μg of peptide, in two injections of 112.5 μg each (450 μl each).

The investigation product according to the invention is preferably injected Subcutaneously (SC) in the region of the outer part of the upper arm intermediate between the elbow and the shoulder. When two separate injections are required, they are preferably administered simultaneously in both arms: for example, injection 1 is on the right arm and injection 2 is on the left arm.

Sequence listing

<110> Yi Msai s corporation (ImCyse s.a.)

Lu Ke Fan De Elston (Vander Elst, luc)

Wenset-Calif. (Carlier, vincent)

<120> peptides and methods for treating diabetes

<130> IMCY-006-PCT

<150> 17160085.1

<151> 2017-03-09

<160> 26

<170> PatentIn version 3.5

<210> 1

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa is Cys, ser or Thr

<400> 1

Cys Xaa Xaa Xaa

1

<210> 2

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa is Cys, ser or Thr

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 2

Xaa Xaa Xaa Cys

1

<210> 3

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> insulin epitope

<400> 3

Leu Ala Leu Glu Gly Ser Leu Gln Lys

1 5

<210> 4

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin epitope

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa is Cys, ser or Thr

<400> 4

Cys Xaa Xaa Xaa Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln

1 5 10 15

Lys

<210> 5

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin epitope

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa is Cys, ser or Thr

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 5

Xaa Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln

1 5 10 15

Lys

<210> 6

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin epitope

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 6

Cys Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln

1 5 10 15

Lys

<210> 7

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + T cell epitope

<220>

<221> MISC_FEATURE

<222> (3)..(4)

<223> Xaa can be any amino acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa is Cys, ser or Thr

<400> 7

His Cys Xaa Xaa Xaa Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

1 5 10 15

Gln Lys

<210> 8

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + T cell epitope

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa is Cys, ser or Thr

<220>

<221> MISC_FEATURE

<222> (3)..(4)

<223> Xaa can be any amino acid

<400> 8

His Xaa Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

1 5 10 15

Gln Lys

<210> 9

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin T cell epitope

<220>

<221> MISC_FEATURE

<222> (3)..(4)

<223> Xaa can be any amino acid

<400> 9

His Cys Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

1 5 10 15

Gln Lys

<210> 10

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin epitope

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa is Cys, ser or Thr

<400> 10

Cys Xaa Xaa Xaa Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

1 5 10 15

Arg Gly

<210> 11

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin T cell epitope

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa is Cys, ser or Thr

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa can be any amino acid

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 11

Xaa Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln

1 5 10 15

Lys Arg Gly

<210> 12

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin T cell epitope

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 12

Cys Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln

1 5 10 15

Lys Arg Gly

<210> 13

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin T cell epitope

<220>

<221> MISC_FEATURE

<222> (3)..(4)

<223> Xaa can be any amino acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa is Cys, ser or Thr

<400> 13

His Cys Xaa Xaa Xaa Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

1 5 10 15

Gln Lys Arg Gly

20

<210> 14

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin T cell epitope

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa is Cys, ser or Thr

<220>

<221> MISC_FEATURE

<222> (3)..(4)

<223> Xaa can be any amino acid

<400> 14

His Xaa Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

1 5 10 15

Gln Lys Arg Gly

20

<210> 15

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif + insulin T cell epitope

<220>

<221> MISC_FEATURE

<222> (3)..(4)

<223> Xaa can be any amino acid

<400> 15

His Cys Xaa Xaa Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

1 5 10 15

Gln Lys Arg Gly

20

<210> 16

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Xaa is Cys, ser or Thr

<400> 16

Cys Pro Tyr Xaa

1

<210> 17

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa is Cys, ser or Thr

<400> 17

Xaa Pro Tyr Cys

1

<210> 18

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 18

Cys Xaa Xaa Cys

1

<210> 19

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<400> 19

Cys Pro Tyr Cys

1

<210> 20

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> peptide comprising insulin T cell epitope

<400> 20

Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly

1 5 10 15

<210> 21

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 21

Ser Xaa Xaa Cys

1

<210> 22

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 22

Thr Xaa Xaa Cys

1

<210> 23

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> MISC_FEATURE

<222> (2)..(3)

<223> CXXS

<400> 23

Cys Xaa Xaa Ser

1

<210> 24

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> Redox motif

<220>

<221> misc

<222> (2)..(3)

<223> Xaa can be any amino acid

<400> 24

Cys Xaa Xaa Thr

1

<210> 25

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> peptide with redox motif and insulin T cell epitope

<400> 25

His Cys Pro Tyr Cys Val Arg Ser Leu Gln Pro Leu Ala Leu Glu Gly

1 5 10 15

Ser Leu Gln Lys Arg Gly

20

<210> 26

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> peptide with redox motif and insulin T cell epitope

<400> 26

His Cys Pro Tyr Cys Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu

1 5 10 15

Gln Lys Arg Gly

20

Claims (11)

1. An isolated immunogenic peptide of up to 30 amino acids in length comprising the sequence Cxx [ CST ] SLQPLALEGSLQK [ SEQ ID No. 4] or [ CST ] xxCSLQPLALEGSLQK [ SEQ ID No. 5], wherein x is any amino acid.

2. The peptide according to claim 1, comprising the sequence cxxcslqplalgslqk [ seq id No. 6].

3. The peptide according to claim 1, comprising the sequence HCxx [ CST ] SLQPLALEGSLQK [ SEQ ID NO:7] or H [ CST ] xxCSLQPLALLEGQK [ SEQ ID NO:8].

4. A peptide according to claim 3 comprising the sequence hcxxcslqplalgslqk [ SEQ ID No. 9].

5. The peptide according to claim 4, consisting of the amino acid sequence HCPYCSLQPLALEGSLQKRG [ SEQ ID NO:26 ].

6. Use of a peptide according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment or prevention of type 1 diabetes.

7. A pharmaceutical composition comprising the peptide of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

8. An in vitro method for generating a population of cytolytic cd4+ T cells for APCs presenting insulin epitopes, comprising the steps of:

-providing peripheral blood cells;

-contacting the cells in vitro with a peptide according to any one of claims 1 to 5; and

-expanding the cells in the presence of IL-2.

9. A population of cytolytic cd4+ T cells for an APC presenting an insulin epitope obtainable by the method of claim 8.

10. Use of a population of cytolytic cd4+ T cells for an APC presenting an insulin epitope obtainable by the method of claim 9 for the preparation of a medicament for the treatment or prevention of type 1 diabetes.

11. Use of the isolated immunogenic peptide of claim 5 in the manufacture of a medicament for treating or preventing type 1 diabetes or for alleviating a symptom of type 1 diabetes.